Crystalline Solids of a METAP-2 Inhibitor and Methods of Making and Using Same

ABSTRACT

The disclosure is in part directed to crystalline forms of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol and variants thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 13/568,476 filed Aug. 7,2012, which is a continuation of PCT/US11/59966 filed Nov. 9, 2011 whichclaims the benefit of and priority to U.S. Ser. No. 61/411,655, filedNov. 9, 2010, the contents of which are hereby incorporated by referencein their entirety.

BACKGROUND

MetAP2 encodes a protein that functions at least in part byenzymatically removing the amino terminal methionine residue fromcertain newly translated proteins, such as, glyceraldehyde-3-phosphatedehydrogenase (Warder et al. (2008) J Proteome Res 7:4807). Increasedexpression of the MetAP2 gene has been historically associated withvarious forms of cancer. Molecules inhibiting the enzymatic activity ofMet AP2 have been identified and have been explored for their utility inthe treatment of various tumor types (Wang et al. (2003) Cancer Res63:7861) and infectious diseases, such as, microsporidiosis,leishmaniasis, and malaria (Zhang et al. (2002) J. Bio Med Sci. 9:34).Notably, inhibition of MetAP2 activity in obese and obese-diabeticanimals leads to a reduction in body weight in part by increasing theoxidation of fat and in part by reducing the consumption of food(Rupnick et al. (2002) Proc Natl Acad Sci USA 99:10730).

6-O-(4-Dimethylaminoethoxy)cinnamoyl fumagillol is a METAP2 inhibitorand is useful in the treatment of e.g., obesity.6-O-(4-Dimethylaminoethoxy)cinnamoyl fumagillol is characterized byformula I:

An amorphous form of a hemioxalate salt of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol has been prepared.However, the existence or preparation of a crystalline form of the freebase of 6-O-(4-Dimethylaminoethoxy)cinnamoyl fumagillol does not appearto be disclosed in the art.

Polymorphism is the ability of a substance to crystalline in more thanone crystal lattice arrangement. Crystallization, or polymorphism, caninfluence many aspects of solid state properties of a drug substance. Acrystalline substance may differ considerably from an amorphous form,and different crystal modifications of a substance may differconsiderably from one another in many respects including solubility,dissolution rate and/or bioavailability. Generally, it is difficult topredict whether or not a given compound will form various crystallinesolid state forms. It is even more difficult to predict the physicalproperties of these crystalline solid state forms. Further, it can beadvantageous to have a crystalline form of a therapeutic agent forcertain formulations, e.g., formulations suitable for subcutaneous use.

SUMMARY

In an embodiment, provided herein is a composition comprising acrystalline form of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol. Acrystalline form of 6-O-(4-dimethylaninoethoxy)cinnamoyl fumagillol,free base, is also provided herein, characterized by a powder X-raydiffraction pattern having a characteristic peak on degrees 2θ at about13.3, or for example, characterized by a powder X-ray diffractionpattern having characteristic peaks in degrees 2θ at 13.3, 17.4, and19.9, or for example, characterized by a powder X-ray diffractionpattern having characteristic peaks in degrees 2θ at 7.1, 13.3, 16.3,17.4, 18.6, 19.4, and 19.9, or for example, characterized, by a powderX-ray diffraction pattern having characteristic peaks in degrees 2θ at5.2, 7.1, 10.4, 13.3, 14.2, 16.3, 17.4, 18.6, 19.4, and 19.9, e.g.,characterized by the crystallization pattern shown in FIG. 1. In someembodiments, the powder X-ray diffraction pattern may be obtained usingCu Kα radiation.

Also provided herein is a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base, haying aspace group of P2₁2₁2₁.

In one embodiment, the crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base, in solutionmay have a ¹H NMR spectrum substantially in accordance with the patternshown in FIG. 6.

Also provided herein is a process for preparing a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, (e.g., form A)comprising:

-   -   a) preparing a solution of 6-O-(4-dimethylaminoethoxy)cinnamoyl        fumagillol, e.g., amorphous 6-O-(4-dimethylaminoethoxy)cinnamoyl        fumagillol, in a solvent. For example, a solvent may be a        secondary ether, e.g., diisopropyl ether, or maybe e.g., a        solvent/antisolvent system, e.g., a toluene:n-heptane mixture,        e.g., with a ratio of n-heptane to toluene of about 4:1;    -   b) heating the solution, e.g., to about 40° C. to about 60° C.,        e.g., to about 50° C., to substantially or completely dissolve        the 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol;    -   c) adjusting the temperature so that solid precipates out of the        solution; and    -   d) isolating the crystalline form of        6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol. Such a process        that includes temperature may comprise cooling the solution to        about 4° C. or less, or to about 2° C. to about 10° C.

A pharmaceutical composition comprising the cyrstalline form providedherein and a pharmaceutically acceptable excipient is contemplated, forexample, a composition that is a suspension formulation suitable forsubcutaneous injection. Provided herein, in an embodiment, is a drugsubstance comprising at least a detectable amount of the providedcrystalline form.

A method of treating obesity in a patient in need thereof is alsoprovided that includes administering to the patient an effective amountof crystalline form provided herein. Also provided herein is a method oftreating obesity in patient in need thereof, comprising subcutaneouslyadministering a composition comprising a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base).

Still another aspect of the invention provides a kit comprising adisclosed a crystalline form.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the X-ray diffraction pattern of Form A.

FIG. 2 is a micrograph of Form A.

FIG. 3 depicts the characterization of Form A by differential scanningcalorimetry (DSC).

FIG. 4 depicts the characterization of Form A bythermogravimetric/differential thermal analysis (TG/DTA).

FIG. 5 depicts the FT-IR spectrum of a disclosed crystal form prepared 1(Form A).

FIG. 6 depicts the NMR spectrum of the dissolved crystal form preparedby Example 1.

FIG. 7 is a X-ray diffraction pattern of Form A.

FIG. 8 is a X-ray diffraction pattern of Form A.

FIG. 9 is a X-ray diffraction patters of Form A.

FIG. 10 is a micrograph of Form A.

FIG. 11 is a X-ray diffraction pattern of Form A.

FIG. 12A is a ORTEP drawing of a Form A crystal; FIG. 12B is acomparison of the X-ray diffraction pattern of Form A at roomtemperature and the pattern calculated from the single-crystal dataobtained at 110 K, and FIG. 12C are the atomic coordinates used toconstruct the ORTEP drawing of FIG. 12A.

FIG. 13 is a micrograph of Form C.

FIG. 14 is the X-ray diffraction pattern of Form C.

FIG. 15 depicts the FT-IR spectrum of Form C.

DETAILED DESCRIPTION

At least in part, this disclosure is directed to crystalline forms of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base). Thedisclosure also provides for a pharmaceutical composition comprisingcrystalline 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base)and a pharmaceutically acceptable carrier. The term “crystalline form”refers to a crystal form or modification can be characterized byanalytical methods such as, e.g., X-ray powder diffraction or Ramanspectroscopy. For example, provided herein is a drug substancecomprising at least a detectable amount of a disclosed crystalline formof 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol.

Provided herein is a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base,characterized by a powder X-ray diffraction pattern, having acharacteristic peak in degrees 2θ at about 13.3 (referred to herein as“Form A”). In one embodiment the crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base) ischaracterized by a powder X-ray diffraction pattern that has acharacteristic peak in degrees 2θ at about 5.2, or is characterized by apowder X-ray diffraction pattern that has a characteristic peak indegrees 2θ at about 7.1, or is characterized by a powder X-raydiffraction pattern that has a characteristic peak in degrees 2θ atabout 10.4, or is characterized by a powder X-ray diffraction patternthat has a characteristic peak in degrees 2θ at about 14.2, or ischaracterized by a powder X-ray diffraction pattern that has acharacteristic peak in degrees 2θ at about 15.5, or is characterized bya powder X-ray diffraction pattern that has a characteristic peak indegrees 2θ at about 16.3, or is characterized by a powder X-raydiffraction pattern that has a characteristic peak in degrees 2θ atabout 17.4, or is characterized by a powder X-ray diffraction patternthat has a characteristic peak in degrees 2θ at about 18.6, or ischaracterized by a powder X-ray diffraction pattern that has acharacteristic peak in degrees 2θ at about 19.4, or is characterized bya powder X-ray diffraction pattern that has a characteristic peak indegrees 2θ at about 19.9, or is characterized by a powder X-raydiffraction pattern that has a characteristic peak in degrees 2θ atabout 20.9, or is characterized by a powder X-ray diffraction patternthat has a characteristic peak in degrees 2θ at about 22.6, or ischaracterized by a powder X-ray diffraction pattern that has acharacteristic peak in degrees 2θ at about 24.6. In another embodiment,the crystalline form is characterized by a powder X-ray diffractionpattern having at least one or more characteristic peaks in degrees 2θat about 13.3, 17.4, and 19.9. In a further embodiment, the crystallineform is characterized by a powder X-ray diffraction pattern having atleast one or more characteristic peaks in degrees 2θ at about 7.1, 13.3,16.3, 17.4, 18.6, 19.4, and 19.9. In yet another embodiment, thecrystalline form is characterized by a powder X-ray diffraction patternhaving at least one or more characteristic peaks in degrees 2θ at about5.2, 7.1, 10.4, 13.3, 14.2, 16.3, 17.4, 18.6, 19.4, and 19.9. In someembodiments, the crystalline form is characterized by a powder X-raydiffraction pattern having at least one or more characteristic peaks indegrees 2θ at about 5.2, 7.1, 10.4, 13.3, 14.2, 15.5, 16.3, 17.4, 18.6,19.4, 19.9, 20.9, 22.6, and 24.6. The term “about” in this context meansthat there is an uncertainty in the measurements of the 2θ of ±0.5(expressed in 2θ) or that there is an uncertainty in the measurements ofthe 2θ of ±0.2 (expressed in 2θ). For example, a contemplatedcrystalline form has a powder X-ray diffraction pattern shown in FIG. 1.In one embodiment, the powder X-ray diffraction pattern of thecrystalline form was obtained using Cu Kα radiation. In a furtherexample, a contemplated crystalline form has a ¹H NMR spectrumsubstantially in accordance with the pattern shown in FIG. 6, whereinthe crystalline form is in solution.

Also provided herein is a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base, having aspace group of P2₁2₁2₁.

The crystalline form of Form A 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol has an IR absorption spectrum having at least one or morecharacteristic peaks at about 2971, 2938, 2817, 2762, 1163, 1103, 832cm⁻¹. In this context, the term “about” means that the cm⁻¹ values canvary, e.g., up to ±5 cm⁻¹. A contemplated crystalline form ischaracterized by the IR absorption spectrum shown in FIG. 5. Thecontemplated crystalline form of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol may be characterized by a melting point of about 83° C., forexample, and may be characterized by a differential scanning calorimetryprofile with an endotherm at about 83.1° C. Form A, for example, has asolubility in diisopropyl ether of about 25 mg/mL at room temperature(ca. 20° C.) and about 102 mg/mL at 50° C. The solubility of Form A insolvent (e.g., an aqueous solution that may include a buffer) with a pHgreater or equal to about 8.0 may be less than about 0.2 mg/mL at ca.20° C. Contemplated crystalline forms disclosed herein may besubstantially more stable as compared, for example, to amorphous freebase and/or amorphous hemioxalate salt of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol.

Also provided herein is a process for preparing a crystalline form6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base), e.g., FormA, comprising:

-   -   a) preparing a solution of 6-O-(4-dimethylaminoethoxy)cinnamoyl        fumagillol, e.g., amorphous 6-O-(4-dmethylaminoethoxy)cinnamoyl        fumagillol in a solvent. Such solvents contemplated may include        e.g., a secondary ether, toluene, n-heptane, or a combination of        two or more solvents, and/or a solvent/anti-solvent system;

b) heating the solution to completely dissolve the6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol;

c) adjusting the temperature so that solid precipitates out of thesolution; and

d) isolating the crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol.

In an exemplary embodiment, the secondary ether is diisopropyl ether.Other contemplated solvents include alcohols such as methanol and/orisopropanol, and solvents such as acetone, acetonitrile, cyclohexane,ethyl acetate, n-heptane, methyl ethyl ketone, methyl isobutyl ketone,tetrahydrofuran, toluene, and/or a combination of two or more thereof.For example, in one embodiment the solvent may be a toluene:n-heptanemixture, wherein the ratio of n-heptane to toluene is, for example,about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, or about 1:1. In another example, the solventor solvent/anti-solvent system is selected from ethyl acetate:n-heptane;acetone:n-heptane; or methyl ethyl ketone:n-heptane. Contemplated ratiosof antisolvent to solvent include, for example, about 15:1, about 14:1,about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1,about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, orabout 1:1. In some embodiments, heating the solution comprises heatingthe solution to about 40° C. to about 60° C., e.g., to about 50° C. Inanother embodiment, adjusting the temperature comprises cooling thesolution to about 0° C. to about 10° C., e.g., to about 4° C. In oneembodiment, adjusting temperature comprises cooling the solution toabout 4° C. or less, or to about 2° C. to about 10° C. Such systems maybe used with or without seeding. For example, contemplated processes mayalso include incorporating or seeding a solution with an existingcrystal of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol.

In another embodiment, a different crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base),characterized by a powder X-ray diffraction pattern havingcharacteristic peaks in degrees 2θ at one or more of positions at about6.1 and 18.4 or at about 6.1, 12.2, 12.8 ,12.9, 18.4, 18.6, 19.7, 20.2,24.1, and 24.7, (referred to herein as “Form C”), is provided. The term“about” in this context means for example, that there is an uncertaintyin the measurements of the 2θ of ±0.5 (expressed in 2θ) or even thatthere is an uncertainty in the measurements of the 2θ of ±0.2 (expressedin 2θ). For example, a contemplated crystalline form has a powder X-raydiffraction pattern shown in FIG. 14.

Form C of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol has an IRabsorption spectrum having characteristic peaks at about at least one of831, 894, 1106, 1159, 1249, 1287, 1512, 1602, 1631, and 1707 cm⁻¹. Inthis context, the term “about” means that the cm⁻¹ values can vary, e.g.up to ±5 cm⁻¹. For example, a contemplated crystalline form ischaracterized by the IR absorption spectrum shown in FIG. 15. Thecontemplated crystalline Form C of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol exhibits plate-like morphology. In one embodiment, Form Cconverts or reverts to Form A after, for example, about three days ofstorage at either 5° C. or ambient temperature.

Methods

In certain embodiments, the disclosure provides a method of treating andor ameliorating obesity in a patient in seed thereof by administering aneffective amount of a disclosed crystalline compound, e.g., Form A. Alsoprovided herein are methods for inducing weight loss in a patient inneed thereof, comprising administering a disclosed crystalline compound.

Other contemplated methods of treatment include methods of treating orameliorating an obesity-related condition or co-morbidity, byadministering a crystalline compound disclosed herein to a subject. Forexample, contemplated herein are methods for treating type 2 diabetes ina patient in need thereof and/or method of treating a patient sufferingfrom diabetes, for other contemplated disease or disorders.

Exemplary co-morbidities or other disorders that may be treated by adisclosed compound may include cardiac disorders, endocrine disorders,respiratory disorders, hepatic disorders, skeletal disorders,psychiatric disorders, metabolic disorders, metabolic disorders, andreproductive disorders.

Exemplary cardiac disorders include hypertension, dyslipidemia, ischemicheart disease, cardiomyopathy, cardiac infarction, stroke, venousthromboembolic disease and pulmonary hypertension. Exemplary endocrinedisorders include type 2 diabetes and latent autoimmune diabetes inadults. Exemplary respiratory disorders include obesity-hypoventilationsyndrome, asthma, and obstructive sleep apnea. An exemplary hepaticdisorder is nonalcoholic fatty liver disease. Exemplary skeletaldisorders include back pain and osteoarthritis of weight-bearing joints.Exemplary metabolic disorders include Prader-Willi Syndrome andpolycystic ovary syndrome. Exemplary reproductive disorders includesexual dysfunction, erectile dysfunction, infertility, obstetriccomplications, and fetal abnormalities. Exemplary psychiatric disordersinclude weight-associated depression and anxiety.

In particular, in certain embodiments, the disclosure provides a methodof treating the above medical indications comprising administering to asubject in need thereof a therapeutically effective amount of a compounddescribed herein. In certain other embodiments, a method of treatingobesity in patient in need thereof is provided, comprisingsubcutaneously administering a composition comprising a crystalline formof 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol.

Obesity or reference to “overweight” refer to an excess of fat inproportion to lean body mass. Excess fat accumulation is associated withincrease in size (hypertrophy) as well as number (hyperplasia) ofadipose tissue cells. Obesity is variously measured in terms of absoluteweight, weight:height ratio, distribution of subcutaneous fat, andsocietal and esthetic norms. A common measure of body fat is Body MassIndex (BMI). The BMI refers to the ratio of body weight (expressed inkilograms) to the square of height (expressed in meters). Body massindex may be accurately calculated using either of the formulas:weight(kg)/height²m²) (SI) or703×weight(lb)/height²(in²) (US).

In accordance with the U.S. Centers for Disease Control and Prevention(CDC), an overweight adult has a BMI of 25 kg/m² to 29.9 kg/m², and anobese adult has a BMI of 30 kg/m² or greater. A BMI of 40 kg/m² orgreater is indicative of morbid obesity or extreme obesity. Obesity canalso refer to patients with a waist circumference of about 102 cm formales and about 88 cm for females. For children, the definitions ofoverweight and obese take into account age and gender effects on bodyfat. Patients with differing genetic background may be considered“obese” at a level differing from the general guidelines describedabove.

The crystalline compounds disclosed herein can be used as a medicamentor pharmaceutically acceptable composition, e.g., in the form ofpharmaceutical preparations for enteral, parenteral, or topicaladministration, and the contemplated methods disclosed herein mayinclude administering enterally, parenterally, or topically a disclosedcrystalline compound, or a composition comprising or formed from such adisclosed crystalline compounds. For example, the disclosed crystallineForm A may be capable of controlling one or more pharmacokineticproperties (e.g., a longer or shorter release profile) when administeredby a certain route (e.g., subcutaneous) or in a certain formulationhaving the amorphous form. In one embodiment, a disclosed crystallineform, e.g., Form A, may afford substantial reproducibility from oneformulation to another.

Compositions

Another aspect of the disclosure provides pharmaceutical compositionscomprising compounds as disclosed herein formulated together with apharmaceutically acceptable carrier. In particular, the presentdisclosure provides pharmaceutical compositions comprising compounds asdisclosed herein formulated together with one or more pharmaceuticallyacceptable carriers. These formulations include those suitable for oral,rectal, topical, buccal, ocular, parenteral (e.g., subcutaneous,intramuscular, intradermal or intravenous) rectal, vaginal, or aerosoladministration, although the most suitable form of administration in anygiven case will depend on the degree and severity of the condition beingtreated, and on the nature of the particular compound being used. Forexample, disclosed compositions may be formulated as a unit dose, and/ormay be formulated for oral or subcutaneous administration.

Exemplary pharmaceutical compositions of this invention may be used inthe form of a pharmaceutical preparation, for example, in solid,semisolid or liquid form, which contains one or more of the compound ofthe invention, as an active ingredient, in admixture with an organic orinorganic carrier or excipient suitable for external, enteral orparenteral applications. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use. The active objectcompound is included in the pharmaceutical composition in an amountsufficient to produce the desired effect upon the process or conditionof the disease.

For preparing solid compositions such as tablets, the principal activeingredient may be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the compositions maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules.

In solid dosage forms, for oral administration (capsules, tablets,pills, dragees, powders, granules and the like), the subject compositionis mixed with one or more pharmaceutically acceptable carriers, such assodium citrate or dicalcium phosphate, and/or any of the following: (1)fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the subject compositionmoistened with an inert liquid diluent. Tablets, and other solid dosageforms, such as dragees, capsules, pills and granules, may optionally bescored or prepared with coatings and shells, such as enteric coatingsand other coatings well known in the pharmaceutical-formulating art.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. Liquid dosage forms for oraladministration include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the subject composition, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, cyclodextrins and mixtures thereof.

Suspensions, in addition to the subject composition, may containsuspending agents, seen as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bestonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing a subject composition withone or more suitable non-irritating excipients or carriers comprising,for example, cocoa butter, polyethylene glycol, a suppository wax or asalicyclate, and which is solid at room temperature, but liquid at bodytemperature and, therefore, will melt in the body cavity and release theactive agent.

Dosage forms for transdermal administration of a subject compositionincludes powders, sprays, ointments, pastes, creams, lotions, gels,solutions, patches and inhalants. The active component may be mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to asubject composition, excipients, such as animal and vegetable fats,oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays may contain, in addition to a subject composition,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of thesessubstances. Sprays may additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Compositions and compounds of the present invention may alternatively beadministered by aerosol. This is accomplished by preparing an aqueousaerosol, liposomal preparation or solid particles containing thecompound. A non-aqueous (e.g., fluorocarbon propellant) suspension couldbe used. Sonic nebulizers may be used because they minimize exposing theagent to shear, which may result in degradation of the compoundscontained in the subject compositions. Ordinarily, an aqueous aerosol ismade by formulating an aqueous solution of suspension of a subjectcomposition together with conventional pharmaceutically acceptablecarriers and stabilizers. The carriers and stabilizers vary with therequirements of the particular subject composition, but typicallyinclude non-ionic surfactants (Tweens, Pluronics, or polyethyleneglycol), innocuous proteins like serum albumin, sorbitan esters, oleicacid, lecithin, amino acids such as glycine, buffers, salts, sugars orsugar alcohols. Aerosols generally are prepared from isotonic solutions.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise a subject composition in combination with one ormore pharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspension or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacterostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate and cyclodextrins. Proper fluidity may be maintained,for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants. For example, crystalline forms providedherein may be milled to obtain a particular particle size, and in atleast some embodiments, such crystalline forms may remain substantiallystable upon milling.

For example, provided herein is a composition suitable for subcutaneousadministration, comprising a suspension of the disclosed crystallineform. Subcutaneous administration can be advantageous over intravenousadministration, which typically requires a doctor visit, and can be morepainful and invasive. A typical dose of the crystalline compound, whenadministered to a patient, may be about 1 mg to about 8 mg of compound.In an embodiment, disclosed herein is a pharmaceutically acceptablecomposition formed from a disclosed crystalline form, e.g., by mixing acrystalline form with an excipient and/or a solvent.

Kits

In one embodiment, a kit for treating obesity or other contemplateddisorder is provided. For example, a disclosed kit comprises a disclosedcrystalline compound, e.g. a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base, e.g., FormA, for example, disposed in an e.g. first container. In someembodiments, a kit may further include a pharmaceutically acceptableexcipient, disposed in e.g. a second container. Such contemplated kitsmay include written instructions describing preparation of apharmaceutical composition suitable for administration to a patient fromthe crystalline form. For example, the written instructions may describepreparing a pharmaceutically acceptable form for patient administrationby e.g. mixing an expicient and a crystalline compound disclosed herein.Disclosed kits may further comprise written instructions describing howto administer the resulting composition to the patient.

EXAMPLES

The compounds described herein can be prepared in a number of ways basedon the teachings contained herein and synthetic procedures known in theart. The following non-limiting examples illustrate the disclosedinventions.

Example 1

Crystalline, Form A material of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol was prepared as follows:

Approximately 423 mg of amorphous gum/oil-like6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol free base compound wasdissolved in ca. 6 mL of diisopropylether (IPE). The solution wasallowed to stir for ca. 24 hours at ambient temperature (18-22° C.)during which time solid precipitated. The resulting solid was isolatedby filtration and dried under vacuum at ambient for ca. 4 hours (yield35.8%).

X-ray powder diffraction (XRPD) analysis was conducted on the solidcrystals (Form A). XRPD analysis was carried out on a Siemens D5000,scanning the samples between 3 and 30 or 50°2θ. For samples<100 mg, ca.5 mg of sample was gently compressed onto a glass substrate which wasinserted into a plastic sample holder. For samples>100 mg, ca. 100 mg ofsample was gently compressed into a plastic sample holder, so that thesample surface was smooth and just above the level of the sample holder.The sample was then loaded into the diffractometer running in reflectionmode and analyzed, using the following experimental conditions, seen inTable 1 below.

TABLE 1 Raw Data Origin Siemens-binary V2 (.RAW) Start Position [°2Th.]3.0000 End Position [°2Th.] 30.000 or 50.000 Step Size [°2Th.] 0.0200Scan Step Time [s] 0.8 Scan Type Continuous Offset [°2Th.] 0.0000Divergence Slit Type Fixed Divergence Slit Size [°] 2.0000 SpecimenLength [mm] various Receiving Slit Size [mm] 0.2000 MeasurementTemperature [° C.] 20.00 Anode Material Cu K-Alpha1 [Å] 1.54060 K-Alpha2[Å] 1.54443 K-Beta [Å] 1.39225 K-A2/K-A1 Ratio 0.50000 (nominal)Generator Settings 40 mA, 40 kV Diffractometer Type D5000 DiffractometerNumber 0 Goniometer Radius [mm] 217.50 Incident Beam Monochromator NoDiffracted Beam Monochromator (Graphite) Spinning No

The XRPD is shown in FIG. 1. Characteristic peaks include one or more ofthe peaks shown in Table 2, below.

TABLE 2 Pos. [°2Th.] Height [cts] d-spacing [Å] Rel. Int. [%] 5.2216879.97 16.92464 38.74 7.1328 1614.46 12.39351 71.08 8.4170 68.5210.50516 3.02 10.3980 1371.44 8.50784 60.38 13.2602 2271.45 6.67717100.00 14.2394 1328.46 6.22010 58.49 14.9084 906.94 5.94247 39.9315.5184 1004.89 5.71023 44.24 15.7074 710.54 5.64192 31.28 16.32121491.01 5.43113 65.64 17.4000 2139.83 5.09673 94.21 18.6247 1628.644.76426 71.70 19.4797 1454.94 4.55704 64.05 19.9991 1691.63 4.4398674.47 20.5602 710.33 4.31993 31.27 20.8627 1054.54 4.25797 46.43 21.0382624.42 4.22285 27.49 21.9610 557.90 4.04744 24.56 22.6008 1083.173.93430 47.69 23.3508 755.63 3.80961 33.27 23.9357 559.19 3.71782 24.6224.5704 1098.96 3.62320 48.38 25.4387 240.68 3.50146 10.60 26.1594243.27 3.40661 10.71 26.6610 598.48 3.34364 26.35 27.0969 679.42 3.2881229.91 27.1788 612.15 3.28111 26.95 27.7736 401.98 3.21218 17.70 28.6369293.31 3.11728 12.91 29.0724 260.75 3.07156 11.48 29.3437 171.15 3.043787.54 30.5513 193.45 2.92617 8.52 32.1240 73.64 2.78641 3.24 32.9570111.68 2.71787 4.92 34.1346 107.25 2.62675 4.72 34.8872 145.93 2.571796.42 35.5321 180.47 2.52657 7.95 37.1636 88.30 2.41932 3.89 38.036845.49 2.36577 2.00 39.4407 74.74 2.28473 3.29 40.2350 76.46 2.24145 3.3741.1595 53.90 2.19321 2.37

The presence of birefringence was determined by polarized lightmicroscopy (PLM) rising as Olympus BX50F4 polarising microscope,equipped with a Motic camera, and image capture software (Motic ImagesPlus 2.0). Images were recorded using 20× objective. Approximately, 1 mgof sample was placed onto a microscope slide in each case, as shown inFIG. 2.

The crystalline compound was also characterized by differential scanningcalorimetry. Approximately 5-10 mg of sample was weighed into analuminum DSC pan and sealed with a pierced aluminum lid(non-hermetically), unless specified otherwise. The sample pan was thenloaded into a Seiko DSC6200 (equipped with a cooler) cooled and held at25° C. Once a stable heat-flow response was obtained, the sample andreference were then heated to ca. 280° C. at scan rate of 10° C./min andthe resulting heat flow response monitored. Nitrogen was used as thepurge gas, at a flow rate of 150 cm³/min. The instrument was temperatureand heat-flow calibrated on a weekly basis using an indium referencestandard. Sample analysis was carried out using Muse Measurementsoftware (version 5.4 U) where the temperatures of thermal events werequoted as the onset temperature, measured according to themanufacturer's specifications. Results are depicted in FIG. 3. Allendotherms present in the DSC traces point in the downward direction.

Thermogravimetric/Differential Thermal Analysis (TG/DTA) was alsoconducted. Approximately, 5-10 mg of sample was weighed into analuminium pan and loaded into a simultaneousthermogravimetric/differential thermal analyser (TG/DTA) held at roomtemperature. The sample was then heated at a rate of 10° C./min from 25°C. to 280° C. during which time the change in sample weight was recordedalong with any differential thermal events (DTA). Nitrogen was used asthe purge gas, at a flow rate of 150 cm³/min. The instrument was weightand temperature calibrated on a monthly basis using a 100 mg referenceweight and an indium reference standard, respectively. Sample analysiswas carried out using Muse Measurement software (version 5.4 U). Resultsare depicted in FIG. 4.

Infra-red spectroscopy was carried out on a Bruker Alpha FT-IRSpectrometer. Approximately, 2-20 mg of material was used for theanalysis and samples were either liquid or solid. Spectra were obtainedusing the following parameters: Resolution: 4 cm⁻¹; Background scantime: 16 scans; Sample scan time: 16 scans; Data collection: 4000 to 400cm⁻¹; Result Spectrum:Transmittance:Software: OPUS version 6.5. FIG. 5depicts the IR spectrum of the prepared crystalline compound.

¹H NMR was performed on a Bruker DPX400 NMR spectrometer. Samples wereprepared in deuterated DMSO, and prepared to between 10-20 mg/ml,concentration, and the spectrum is depicted in FIG. 6.

Example 2

Crystallin, Form A material of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol (free base) was scaled up as follows:

Diisopropyl ether (90 mL) was added to a round-bottomed flask (250 mL)containing 11.14 g of amorphous gum/oil-like material. The flask sasthen heated to 50° C. with a condenser attached to the neck of theflask. This allowed for the amorphous material to dissolve. The solutionwas stirred at ca. 300 rpm. After remaining at 50° C. for five minutes,the solution was then cooled at a rate of ca. 1° C./minute whilststirring at ca. 300 rpm. Once the temperature had cooled down to 46° C.,68.2 mg of crystalline material was added to the flask for seeding.After having cooled down to ca. 24° C., solid began to precipitate outof solution and the precipitation continues as the experiment was cooleddown to 4° C. After reaching 4° C., it was held at this temperature forca. 5 minutes. The material was then filtered and allowed to stand onthe filter for 5 minutes in oder to dry. The material was thentransferred to stand on the filter for 5 minutes in order to dry. Thematerial was then transferred to a beaker and placed ina vacuum oven(ca. 600 mbar) at ambient temperature (ca. 20° C.) in order to dryfurther. After remaining in the vacuum oven for 24 hours, the sample wasweighed.

NMR analysis indicated the presence of ca. 2% residual solvent afterdrying for 24 hours. The sample was therefore dried for a further 24hours (i.e., 48 hours of drying in total) under vacuum (ca. 600 mbar) atambient temperature (ca. 20° C.). After the further drying was carriedout, no trace of residual solvent could be identified by NMR analysis.The yield was 80%, and HPLC analysis indicated a purity of greater than99.5%. XPRD is shown in FIG. 7.

Example 3

The grinding of 500 mg of crystalline, Form A material of6-O-(4-dimethyaminoethoxy)cinnamoyl fumagillol was done, as follows:

A sample (ca. 500 mg) from the scale-up of crystalline compound as inExample 2 was placed onto a mortar (Agate material, H: 35 mm, L; 77 mm).The sample was then ground using a pestle (length: 80 mm; grindingdiameter: 17 mm) for approximately 5 minutes. Throughout the grindingprocedure, the sample was allowed to stand for ca. 10 secondsintermittently to ensure that significant heat was not generated. PLMindicated birefrigent material with particle sizes measuring betweenabout 20 μm and 80 μm in length. XRPD analysis indicated that thematerial remained highly crystalline with peak positions consistent withthe un-ground crystalline material (FIG. 8).

Example 4

Crystalline, Form A material of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol (free base) was scaled up as follows:

A 20 mL round-bottom flask was equipped with a stir bar or mechanicalstirrer and a reflux condenser (it is not needed to have the condenserconnected to cold water supply; air cooling is typically enough for thecrystallization purpose). In a separate small vial,6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (1 g) was dissolved inethyl acetate (1 mL). The resulting solution was filtered through a PTFE0.2 um filter into the aforementioned 20 mL round bottomed flask usingnitrogen pressure. The vial was washed with ethyl acetate (0.25) mL),and the resulting solution was filtered through the same PTFE 0.2 umfilter using nitrogen pressure into the flask containing the filtrate.n-Heptane (10 mL) was filtered through the same PTFE 0.2 μm filter usingnitrogen pressure into the flask containing the filtrate (Note:significant precipitation is observed during the addition of n-heptaneto the ethyl acetate solution). The resulting mixture was slowly heatedto about 50-55° C. (Note: complete dissolution is often seen between35-40° C). The solution was slowly cooled to 35° C., at which pointstirring is stopped and seed crystals (1 mg, pulverized) were added. Theinternal temperature of the solution was maintained at about 35° C.without stirring for 3 h (Note: if significant crystal formation on theflask surface was observed, occasional short (about 15 minutes) andstrong agitation strokes were applied to break crystals on the flasksurface). The mixture wax slowly cooled to 20° C. at a rate of 1° C. perhour with no or minimal stirring. The internal temperature of themixture was maintained at about 20° C. for 10-18 h. The product wascollected by filtration as white needle crystals and was washed withn-heptane (0.5 mL), and dried under the filtration vacuum conditions forabout 2 h. The solids were collected onto a pre-weighed petri dish, andthe petri dish was covered and placed into a vacuum oven (21-25° C. at20 mmHg) for more than 18 h to afford crystalline Form A (75-80%).

XRPD analysis indicated that the material was crystalline with a patternconsistent with Form A.

Example 5

Crystalline, Form A material of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol (free base) was scaled up as follows:

A 20 mL round-bottom flask was equipped with a stir bar or mechanicalstirrer and a reflux condenser (it is not needed to have the condenserconnected to cold water supply; air cooling is typically enough for thecrystallization purpose). In a separate small vial,6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (1 g) was dissolved inethyl acetate (1 mL). The resulting solution was filtered through a PTFE0.2 um filter into the aforementioned 20 mL round bottomed flask usingnitrogen pressure. The vial was washed with ethyl acetate (0.25 mL), andthe resulting solution was filtered through the same PTFE 0.2 um filterusing nitrogen pressure into the flask containing the filtrate.n-Heptane (10 mL) was filtered through the same PTFE 0.2 um filter usingnitrogen pressure into the flask containing the filtrate (Note:significant precipitation is observed during the addition of n-heptaneto the ethyl acetate solution). The resulting mixture was slowly heatedto about 50-55° C. (Note: complete dissolution is often seen between35-40° C.). The solution was slowly cooled to 25° C., and thistemperature was maintained with slow stirring for 3 h (Note: whiteprecipitation crashes and stirring speed may need to be adjusted forefficient mixing). The mixture was slowly cooled to 20° C., and theinternal temperature of the mixture was maintained at this temperaturefor 10-18 h. The product was collected by filtration as a white fluffysolid and was washed with n-heptane (0.5 mL), and dried under thefiltration vacuum conditions for about 2 h. The solids were collectedonto a pre-weighed petri dish, and the petri dish was covered and placedinto a vacuum oven (21-25° C. at 20 mmHg) for more than 18 h to affordcrystalline Form A (75-80%).

XRPD analysis indicated that the material was crystalline with a patternconsistent with Form A.

Example 6

Crystalline, Form A material of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol (free base) was scaled up as follows:

A 20 ml round-bottom flask was equipped with a stir bar or mechanicalstirrer and a reflux condenser (it is not needed to have the condenserconnected to cold water supply; air cooling is typically enough for thecrystallization purpose). In a separate small vial,6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (1 g) was dissolved intoluene (about 1 mL). The resulting solution was filtered through a PTFE0.2 um filter into the aforementioned 20 mL round bottomed flask usingnitrogen pressure. The vial was washed with toluene (warm or roomtemperature, 0.25 mL), and the resulting solution was filtered throughthe same PTFE 0.2 um filter using nitrogen pressure into the flaskcontaining the filtrate. n-Heptane (5 mL) was filtered through the samePTFE 0.2 um filter using nitrogen pressure into the flask containing thefiltrate (Note: significant precipitation is observed during theaddition of n-heptane to the toluene solution). The resulting mixturewas slowly heated to about 50-55° C. (Note: complete dissolution isoften seen between 35-40° C.). The solution was slowly cooled to 28° C.,at which time seed crystal (1 mg, pulverized) was added. The internaltemperature of the solution was maintained at about 28 without stirringfor 3 h (Note: if significant crystal formation on the flask surface wasobserved, occasional short (about 15 minutes) and strong agitationstrokes were applied to break crystals on the flask surface). Themixture was slowly cooled to 20° C. at a rate of 1° C. per hour with noor minimal stirring. The internal temperature of the mixture wasmaintained at about 20° C. for 10-18 h. The product was collected byfiltration as white rod crystals and was washed with n-heptane (0.5 mL),and dried under the filtration vacuum conditions for about 2 h. Thesolids were collected onto a pre-weighed petri dish, and the petri dishwas covered and placed into a vacuum oven (21-25° C. at 20 mmHg) formore than 18 h to afford crystalline Form A (65-75%).

XRPD analysis indicated that the material was crystalline with a patternconsistent with Form A.

Example 7

A crystalline version of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol(free base) was scaled up as follows: amorphous gum/oil-like free basematerial from three different vessels was combined into a 500 mL roundbottomed flask as seen in Table 3:

TABLE 3 Weight of sample DIPE added DIPE added for Vessel removedinitially washing out vessel Round bottomed flask 13.94 g 70 ml 20 mlRound bottomed flask 14.16 g 70 ml 20 ml 20 ml Vial  0.46 g  3 ml  2 ml

After the initial addition of diisopropyl ether to each vessel, thethree vessels were heated to 50° C. whilst stirring at ca. 300 rpm andkept at this temperature until the majority of the material appeared tohave dissolved. The solutions from each vessel were then transferred toa 500 mL round bottomed flask. A second addition of diisopropyl etherwas then added to each vessel in order to dissolve the remainingmaterial and wash out the vessels into the 500 mL round bottomed flask.Alter the combination of the material from the three vessels, the 500 mLround bottomed flask contained ca. 28.56 g material dissolved in ca. 185mL diisopropyl ether. The flask was then heated to 50° C. whilststirring at ca. 300 rpm and held at this temperature for approximately10 minutes. This allowed for all material to dissolve completely. Afterremaining at 50° C. for 10 minutes, the solution was then cooled at arate of ca. 1° C./minute whilst stirring at ca. 300 rpm. Once thetemperature bad cooled down to 30° C., 14.8 mg of crystalline materialmade as in, for example, Example 1 was added to the flask for seeding(the crystalline seeding material had been ground for ca. 1 minute usingan agate mortar and pestle, before it was added to the flask as seed).As the cooling continued down to 4° C., solid precipitated out of thesolution until a thick slurry resulted. The flask was held at 4° C. fora further one hour whilst stirring at ca. 300 rpm. The material was thenfiltered and allowed to stand on the filter for approximately 10 minutesin order to dry. The material was then transferred to a beaker andplaced into a vacuum oven (ca. 600 mbar) at ambient temperature (ca. 20°C.) in order to dry further. After remaining in the vacuum oven for 48hours, the sample was weighed. ¹H NMR analysis indicated the presence ofca. 2.4% residual solvent after drying for 48 hours. The sample wastherefore dried for a further 3 days (i.e., 5 days of drying in total)under vacuum (ca. 600 mbar) at ambient temperature (ca. 20° C.). Afterthe further drying was carried out, ¹H NMR analysis indicated thepresence of 1.13% residual solvent. The sample was then dried for afurther 3.5 days (i.e., 8.5 days of drying in total) under vacuum (ca.600 mbar) at 30° C.

¹H NMR analysis was then carried out and no trace of residual solventcould he identified. HPLC analysis indicated purity of greater than99.5%. XPRD is shown in FIG. 9.

Example 8

A crystalline version of 6-O-(4-dimethylaminoethoxyl)cinnamoylfumagillol, free base, was scaled up from fumagillol as follows:

In a 5 L glass reactor, toluene (1.5 L), fumagillol (300 g),1-ethyl-(3-dimethylaminopropyl)-carbodiimide (EDC; 3.75 g (87.9%)),N,N-dimethylaminopyridine (DMAP; 261 g), and4-[(2-N,N-dimethylamino)ethoxy]cinnamic acid (501 g) were added to thereactor in that order at room temperature. The mixture was heated from20° C. to about 45-58° C. over 30 minutes, and stirred at thattemperature for another 1-3 h until the reaction was complete. Reactioncompletion was monitored by thin-layer chromatography(dichloromethane:methanol (4:1), silica plate, anisaldehydevisualization) with less than 1% of fumagillol present (Note: thereaction typically requires between 2-3 h for completion).

After the reaction was confirmed to be completed, the mixture was cooledto 20-25° C. over 35 minutes and toluene (1:5 L) was added. Theresulting mixture was filtered through a celite pad (300 g) to removeall undissolved materials and the celite pad was washed with toluene(3.0 L). The combined filtrate (6.85 L) was quantitatively analyzed byHPLC (520 g (97%) of the desired product was estimated present in thefiltrate solution).

The toluene filtrates were washed pH 4.0-4.5, 250 mM ammonium acetatebuffer solution (2 washes, 4.5 L per wash). The ammonium acetate buffersolution was prepared by dissolving ammonium acetate (174 g) in purifiedwater (9 L) and adjusting the pH by addition of acetic acid (283 g).After confirming the removal of most of the DMAP and cinnamic acid (thinlayer chromatography analysis (dichloromethane: methanol (4:1),anisaldehyde visualization)), the organic phase was washed with 5%NaHCO₃ (1.5 L) and purified water (1.5 L). An HPLC analysis wasperformed and no DMAP was detected.

Activated carbon (30 g, Nuchar SA-20) was added to the toluene solutionand the mixture was stirred for 20 minutes. The activated carbon wasremoved by filtering the suspension through a celite pad (300 g) over 20minutes, and the filtrate solution was filtered through a 0.2 um filter(Waters, Catalog No: 186003524) over another 20 minutes. The toluenesolution was concentrated using a rotary evaporator in vacuo (bath,temperature=35-40° C. 15-25 mbar), and the ¹H HMR of the concentrate wastaken to determine the residual toluene as 15.3%.

To the concentrate, n-heptane (1.0 L) was added and the resultingmixture was reconcentrated in vacuo (bath temperature=35-40° C., 15-25mbar) over 25 minutes (product turned into a lumpy solid). The residualtoluene in the concentrate was determined as 0% from ¹H NMR analysis.

To this concentrate was added toluene (0.3 L filtered through a 0.2 umfilter) and n-heptane (1.2 L filtered through a 0.2 um filter) and theresulting mixture was slowly heated to 40-51° C. over 40 minutes,resulting in complete dissolution of the solid. The mixture was slowlycooled to 25-36° C. and 45 mg of Form A seed crystal was added. Themixture remained at room temperature without agitation for 10-25 hours.

The product was collected by filtration and the filter cake was washedwith n-heptane (300 mL of 0.2 um filtered), and dried at between 28-30°C. under vacuum (0.2-0.3 inch Hg) for 24 hours to provide a crystallineform of 6-O-(4-dimetlylaminoethoxy)cinnamoyl fumagillol, free base (375g, 70.6:%), with 98-99% HPLC purity (The filtrate was concentrated toprovide the filtrate concentrate (117 g), which had 80.9% purity by anHPLC analysis).

XRPD analysis indicated that the material was crystalline with a patternconsistent with Form A.

Example 9

Recrystallization of a crystalline version of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillo (free base) was performedas follows:

A 250 mL round-bottom flask was charged with crystalline6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (free base; 19 g).Toluene (ca. 19 mL) was added and the reaction was slowly heated (ca. 1°C./minute) on a magnetic hotplate stirred (with heating mantle) to ca.55° C., whilst stirring (oval magnetic stirrer bar, length: 2.5 cm) atca 150 rpm. After complete dissolution, heptane (ca., 171 mL, pre-heatedto ca. 55° C.) was slowly added and solid material began to immediatelyprecipitate out of solution. After 10 minutes of stirring, theprecipitated solid had dissolved, however a small amount of yellow gumwas present. The solution was transferred to a different round-bottomflask (250 mL, pre-heated to ca. 55° C.) in order to remove the gum. Thetransferred solution was allowed to stir slowly (ca. 150 rpm) in the newflask for ca. 5 minutes before the hotplate was turned off and thereaction naturally cooled from 55° C. down to ambient (ca., 22° C.).Solid material crystallized out of solution at ca. 28° C. After coolingto ambient (ca. 22° C.), slow stirring (ca. 150 rpm) of the slurry wascontinued for a further 3 hours. After 3 hours, the solid was filteredusing a Büchner funnel )diameter: 7.7 cm) and Büchner filter flask (500mL) connected to a small diaphragm pump. Double filter paper was used inthe filter (filter paper diameter 5.5 cm). The material was allowed todry on the filter for ca. 10 minutes. The solid material was then placedinto a crystallization dish with a large surface area (diameter ca. 25mbar, absolute pressure reading) at ambient (ca., 22° C.) forapproximately seven days to provide a crystalline form of6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base (ca., 15.1 g,79.4%).

XRPD analysis indicated that the material was crystalline with a patternconsistent with Form A.

Example 10

Crystalline material of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol(Form A) suitable for X-ray determination was prepared utilizing thesolvent-antisolvent by vapor diffusion approach, as follows:

A filtered solution of Form A with a concentration of 100 mg/mL, wasprepared by stirring a sample of Form A (see FIGS. 10 and 11) in theappropriate amount of methyl-t-butyl ether at ambient temperature andthen filtering the solution through a 0.7 μm glass fiber filter into a1.2 mL vial insert. At this time, the filtered solution was exposed tovapors of pentane, resulting in the formation of crystalline material,which was submitted for single crystal structure determination. Thesingle crystal structure determination procedure was conducted, asfollows:

The single-crystal sample of Form A was mounted on a Mitegen polymidemicromount with a small amount of Paratone N oil. All X-ray measurementswere made on a Bruker-Nonius Kappa Axis X8 Apex2 diffractometer at atemperature of −163° C. The unit cell dimensions were determined from asymmetry constrained fit of 9994 reflections with 4.76°<2θ<55.5°. Thedata collection strategy was a number of ω and φ scans which collecteddata up to 59.34° (2θ). The frame integration was performed using SAINT(Bruker-Nonius, SAINT version 2009.9, 2009, Bruker-Nonius, Madison, Wis.53711, USA). The resulting raw data was scaled and absorption correctedusing a multi-scan averaging of symmetry equivalent data using SADABS(Bruker-Nonius, SADABS version 2009.9, 2009, Bruker-Nonius, Madison,Wis.).

The crystal structure was solved by direct methods using the XS program(Bruker-AXS, XS version 2009.9, 2099, Bruker-AXS, Madison, Wis. 53711,USA). All non-hydrogen atoms were obtained from the initial solution.The hydrogen atoms were introduced at idealized positions and wereallowed to ride on the parent atom. The C3 atom site was disordered over2 positions. The alternate position was designated C3′. The normalizedoccupancy for the primary position refined to a value of 0.698(10). Theabsolute structure could not be determined from the diffraction data.The absolute configuration of C14 was set to the absolute configuration(R) the corresponding atom (C6) reported in the structure of fumagillol(Haláz, J. et. al. Tetrahedron, 2000, 56, 10081.). All otherstereocenters were set relative to that assignment. The structural modelwas fit to the data using full matrix least-squares based on F². Thecalculated structure factors included corrections for anomalousdispersion from the usual tabulation. The structure was refined usingthe XL program from SHELXTL (Bruker-AXS, XL version 2009.9, 2009,Bruker-AXS, Madison, Wis. 53711, USA), graphic plots were produced usingthe NRCVAX crystallographic program suite.

The ORTEP drawing for the single crystal determination is shown in FIG.12A. The summary of the crystal data is seen in Table 5, below.

TABLE 5 Formula C₂₉H₄₁NO Formula Weight (g/mol) 499.63 CrystalDimensions (mm) 0.43 × 0.15 × 0.06 Crystal Color and Habit colourlessprism Crystal System orthorhombic Space Group P 2₁ 2₁ 2₁ Temperature, K110 a, Å 6.2327(16) b, Å 13.118(4) c, Å 33.857(9) α, ° 90.00 β, ° 90.00γ, ° 90.00 V, Å³ 2768.2(13) Number of reflections to determine finalunit cell 9994 Min and Max 2θ for cell determination, ° 4.76, 55.5 Z 4F(000) 1080 ρ (g/cm) 1.199 λ, Å, (MoKα) 0.71073 μ, (cm⁻¹) 0.083Diffractometer Type Bruker-Nonius Kappa Axis X8 Apex2 Scan Type(s) omegaand phi scans Max 2θ for data collection, ° 59.34 Measured fraction ofdata 0.991 Number of reflections measured 56971 Unique reflectionsmeasured 4338 R_(merge) 0.0435 Number of reflections included inrefinement 4338 Cut off Threshold Expression >2sigma(I)

The data for the crystal structure of the ORETP drawing of FIG. 12A isshown in FIG. 12C. A comparison of the X-ray diffraction pattern of FormA at room temperature and the pattern calculated from the single-crystaldata obtained at 110 K is shown in FIG. 12B.

Example 11

Crystalline, Form C material of 6-O-(4-dimethylaminoethoxy)cinnamoylfumagillol was prepared as follows:

Amorphous material was prepared by dissolving6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol (20 mg) in methanol (0.5mL), and placing the resulting solution in a centrifuge evaporator for 4h. The amorphous phase can be detected using Raman spectroscopy, whereinthe amorphous material displayed characteristic peaks at 1633 and 1703cm⁻¹, while Form A displayed related peaks at 1627 and 1700 cm⁻¹.

A sample of amorphous material of 6-O-(4-dimethylaminoethyoxy)cinnamoylfumagillol was exposed to vapors of neat trichloroethane at ambienttemperature. The amorphous form readily deliquesced. The deliquescedsample was stored in a cold environment and evaporated to dryness usinga Genevac centrifuge evaporator. The sample was then scaled, submergedin dry ice for ca. 15 minutes and stored in a freezer (ca. 25° C.). Thesample remained glassy during storage in the freezer (−20° C., 9 days),and was then stored at 5° C. (9 days), resulting in crytsalline, Form C(see FIG. 13 for micrograph). A portion of the Form C sample was left atambient temperature. The sample at ambient temperature, as well as thesample stored at 5° C., converted to Form A after three days. Form C wasobserved to be metastable relative to Form A.

X-ray powder diffraction (XRPD) analysis was conducted on the solidcrystals (Form C). XRPD analysis was carried out on a Bruker D8Discovery diffractrometer with a HI-STAR GADDS detector or on aPANalytical X'Pert Pro diffractometer on a Si zero-background wafers.All diffractograms were collected using a monochromatic Cu Kα (45 kV/40mA) radiation and a step size of 0.02°2θ. The XRPD is shown in FIG. 14.The XRPD patient of Form C does not show any of the characteristic peaksof Form A, and is though to be phase-pure.

Characteristic XRPD peaks include one or more of the peaks shown inTable 4, below.

TABLE 4 Position d-spacing [°2Th.] [Å] 18.4 4.8 6.1 14.6 12.9 6.8 12.86.9 18.6 4.8 12.2 7.2 19.7 4.5 20.2 4.4 24.1 3.7 24.7 3.6

Infra-red spectroscopy was carried out on a Nicolet 6700 spectrometer(Thermo Electron) equipped with a DTGS detector and a Durascope. Spectrawere obtained using the following parameters: 4 cm⁻¹ resolution, 64scans, using Happ-Genzel apodization function and 2-level zero-filling.

FIG. 15 depicts the IR spectrum of the prepared crystalline, form Ccompound. As seen in FIG. 15, the IR spectrum of Form C shows peakshifts relative to Form A. For example, in the carbonyl region Form Cshows a peak at 1707 cm⁻¹, while Form A shows a corresponding peak at1700 cm⁻¹. In another example, Form C shows a peak at 894 cm⁻¹, whileForm A does not show a similar peak in the fingerprint region.

Characteristic IR peaks include one or more of the peaks shown in Table5, below.

TABLE 5 FT-IR Absorption Bands, cm⁻¹ 1159 1602 1707 1512 1249 831 12871106 1631 894

Raman spectroscopy was conducted utilizing a Nicolet NXR9650 or NXR 960spectrometer (Thermo Electron) equipped with 1064 nm Nd:YVO₄ excitationlaser, InGaAs and liquid-N₂ cooled Ge detectors, and a MicroStage. Allspectra were acquired at 4 cm-1 resolution, 64-128 scans, usingHapp-Genzel apodization function and 2-level zero-filling.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety forall purposes as if each individual publication or patent wasspecifically and individually incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification. The full scope of the inventionshould be determined by reference to the claims, along with their fullscope of equivalents, and the specification, along with such variations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

What is claimed is: 1-21. (canceled)
 22. A kit comprising (i) acrystalline form of 6-O-(4-dimethylaminoethoxy)cinnamoyl fumagillol,free base, characterized by a powder X-ray diffraction pattern having acharacteristic peak in degrees 2θ at about 13.3, 17.4, and 19.9,disposed in an first container; and (ii) a pharmaceutically acceptableexcipient, disposed in a second container.
 23. The kit of claim 22,wherein the crystalline form is characterized by a powder X-raydiffraction pattern having characteristic peaks in degrees 2θ at about7.1, 13.3, 16.3, 17.4, 18.6, 19.4, and 19.9.
 24. The kit of claim 22,wherein the crystalline form is characterized by a powder X-raydiffraction pattern having characteristic peaks in degrees 2θ at about5.2, 7.1, 10.4, 13.3, 14.2, 16.3, 17.4, 18.6, 19.4, and 19.9.
 25. Thekit of claim 22, wherein the crystalline form has the powder X-raydiffraction pattern shown in FIG.
 1. 26. The kit of claim 22, whereinthe powder X-ray diffraction pattern was obtained using Cu Kα radiation.27. The kit of claim 22, wherein the crystalline form has a space groupof P2₁2₁2₁.
 28. The kit of claim 22, wherein the first container has 1mg to about 8 mg of the crystalline form.
 29. The kit of claim 22,wherein the pharmaceutically acceptable excipient comprises water.
 30. Akit comprising (i) a crystalline form of6-0-(4-dimethylaminoethoxy)cinnamoyl fumagillol, free base,characterized by a powder X-ray diffraction pattern having acharacteristic peak in degrees 2θ at about 13.3, 17.4, and 19.9,disposed in an first container; and (ii) one or morepharmaceutically-acceptable sterile isotonic aqueous solutions, disposedin a second container.